Despite the intrinsic ability of bone to heal, there are numerous clinical circumstances where bone healing is defective. One such circumstance occurs with the loss of large segments of bone, leading to critical sized defects that will not heal spontaneously. Various growth factors, including bone morphogenetic protein-2 (BMP-2), the focus of this project, stimulate new bone formation, but clinical application is hindered by delivery problems. This project is based upon the hypothesis that these problems can be overcome, and clinical utility enhanced, by delivering the gene encoding the growth factor, rather than the growth factor itself. Here, a gene transfer approach to delivering BMP-2 to critical sized segmental defects will be evaluated in a rat model. An adenovirus vector (Ad.BMP-2) will be used for this purpose. Two gene transfer strategies will be compared for safety and efficacy. In the first, virus is injected directly into the defect (in vivo delivery). In the other, autologous marrow is removed intraoperatively, mixed with vector, allowed to clot, and inserted into the defect (gene plug delivery). Healing will be assessed radiographically and by micro-computed tomography, dual-energy X-ray absorptiometry, histology, histomorphometry and by biomechanical testing. Safety will be evaluated by determining the tissue distribution of the transgenes, by histopathological examination of organs, and by immunologic criteria. As part of the safety evaluation, the effects of prolonged over-expression in athymic animals will also be determined. This information will be collated, thus permitting an informed, evidenced-based selection of the gene transfer system that is most effective and safest for possible future human application. The molecular biology of bone healing induced by the selected modality will then be further investigated. The results of this project will serve as the basis for planning early phase clinical protocols, or will have identified the critical, key matters that future research needs to address prior to developing such protocols. [unreadable] [unreadable]